1 Field of the Invention
Significant morbidity and mortality are associated with infectious diseases. More rapid and accurate diagnostic methods are required for better monitoring and treatment of disease. Molecular methods using DNA probes, nucleic acid hybridizations and in vitro amplification techniques are promising methods offering advantages to conventional methods used for patient diagnoses.
A method for the enzymatic amplification of specific segments of DNA known as the polymerase chain reaction (PCR) method has been described. This in vitro amplification procedure is based on repeated cycles of denaturation, oligonucleotide primer annealing, and primer extension by thermophilic polymerase, resulting in the exponential increase in copies of the region flanked by the primers. The different PCR primers, which anneal to opposite strands of the DNA, are positioned so that the polymerase catalyzed extension product of one primer can serve as a template strand for the other, leading to the accumulation of a discrete fragment whose length is defined by the distance between the 5xe2x80x2 ends of the oligonucleotide primers.
Another method has also been described for amplifying nucleic acid sequences. This method is referred to as single primer amplification. The method provides for the amplification of a target sequence that possesses a stem-loop or inverted repeat structure where the target sequence is flanked by relatively short complementary sequences. Various methods for creating such a target sequence in relation to the presence of a polynucleotide analyte to be detected have also been described.
The amplification methods described above require that samples suspected of having a specific nucleotide sequence be heated at about 95xc2x0 C. and then be repetitively thermally cycled between one or two lower temperatures and about 95xc2x0 C. The higher temperatures denature duplexes and the lower temperatures permit hybridization of the primer and chain extension.
The above methods are extremely powerful techniques for high sensitivity detection of target DNA molecules present in very small amounts. The correlation between the number of original target DNA molecules and the number of specifically amplified products is influenced by a number of variables. Minor variations in buffer or temperature conditions can greatly influence reaction-to-reaction amplification efficiencies. Further, clinical samples of DNA targets can contain inhibitory factors that can suppress enzymatic amplification. In addition, such clinical samples also contain irrelevant DNA, which can be present in very large amounts relative to the target DNA molecules.
The above amplification methods suffer from interference caused by random partial hybridization of primers used in such amplification to irrelevant DNA, i.e., DNA that is not target DNA and to which the primers bind non-specifically or non-selectively. A competition between target DNA and irrelevant DNA for the enzyme and the primer thus is created. As a result the efficiency of the amplification of the target DNA molecules is decreased. At best this leads to difficulty in distinguishing amplified target DNA from amplified irrelevant DNA. The amplification of irrelevant DNA to any substantial degree can interfere with specific amplification of target DNA to prevent detection of the target DNA completely.
One approach for this problem is to avoid chain extension of low temperature non-specifically hybridized primers by heating the reaction mixture to 95xc2x0 C. prior to adding a critical reagent such as a polymerase enzyme or magnesium that is required to activate the polymerase. This can be accomplished by using a wax layer to separate the various reaction components until a high temperature is reached. Alternatively, an inhibitory antibody against the polymerase can be added at low temperature. The antibody denatures at elevated temperature and allows the enzyme to become reactivated. Another approach involves the use of AmpliTaq Gold(copyright) enzyme as the polymerase in PCR reactions.
Another method involving chain extension of an oligonucleotide primer is a method for the detection of differences in nucleic acids described in U.S. Patent Application Serial No. 60/009,289, the disclosure of which is incorporated herein by reference. Briefly, the branch migration method detects a difference between two related nucleic acid sequences. In the method, if there is a difference between the two related nucleic acid sequences, a stable quadramolecular complex is formed comprising both of the nucleic acid sequences in double stranded form. Usually, the complex comprises a Holliday junction. Both members of at least one pair of non-complementary strands within the complex have labels. The association of the labels as part of the complex is determined as an indication of the presence of the difference between the two related sequences. The method may be employed for detecting the presence of a mutation in a target nucleic acid sequence or for detecting the presence of a target nucleic acid sequence.
In the above method for the detection of differences between two related DNA sequences, non-specific priming can be a problem for mutation detection by inhibition of DNA branch migration. All amplification products incorporate the xe2x80x9ctailxe2x80x9d sequences of the reverse primers and hence are able to participate in the formation of four-stranded DNA complexes with both specific PCR products and with each other. Since the sequences on both sides of the junction are completely different from each other, such complexes never undergo strand separation by branch migration and thus generate non-specific signal. One approach to alleviate this problem is to use a two-step PCR procedure or nested PCR. It is highly desirable, however, to perform the above method using a single PCR reaction with just one set of primers.
A method for avoiding the above problems that is inexpensive and more controllable than the approaches mentioned above is desirable.
2. Description of the Related Art
A simplified hot start PCR using AmpliTaq Gold(copyright) enzyme is discussed by Birch, et al., Nature (1996) 381:445-446.
A hot start procedure using wax beads is disclosed by Chou, et al., Nucleic Acids Research (1992) 20:1717-1723.
W. B. Barnes discusses PCR amplification of up to 35-kb DNA with high fidelity and high yield from xcex-bacteriophage templates in Proc. Nat. Acad. Sci. USA (1994) 91:2216-2220.
A process for amplifying, detecting and/or cloning nucleic acid sequences is disclosed in U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188 and 5,008,182. Sequence polymerization by polymerase chain reaction is described by Saiki, et al., (1986) Science, 230: 1350-1354. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase is described by Saiki, et al., Science (1988) 239:487.
U.S. Pat. No. 5,508,178 (Rose, et al.) describes nucleic acid amplification using a single polynucleotide primer. U.S. patent application Ser. No. 08/140,349 filed Oct. 20, 1993 (Laney, et al.), describes methods of introducing defined sequences at the 3xe2x80x2-end of polynucleotides. The disclosures of these references are incorporated herein by reference.
Amplification of nucleic acid sequences using oligonucleotides of random sequence as primers is described in U.S. Pat. No. 5,043,272 (Hartley).
Nickel, et al., J. Biol. Chem. (1992) 267:848-854 describes interactions of azidothymidine triphosphate with the cellular DNA polymerases xcex1, xcex4 and xcex5 and with DNA primase.
EP 0 439 182 (Backman, et al.) discusses methods of amplifying target nucleic acids applicable to both polymerase and ligase chain reactions.
In its broadest aspect the present invention relates to a method for controlling the concentration of an oligonucleotide primer in a reaction medium for conducting a reaction in which the oligonucleotide primer is extended along a polynucleotide template in the presence of reagents for carrying out such extension, e.g., a nucleotide polymerase and nucleoside triphosphates. A reaction medium is provided that comprises a modified oligonucleotide together with reactants for extending the oligonucleotide primer along the polynucleotide template. The modified oligonucleotide has a degradable 3xe2x80x2-end that is substantially incapable of being extended along the polynucleotide template. Degradation of the 3xe2x80x2-end of the modified oligonucleotide results in the formation of the oligonucleotide primer. The reaction medium is subjected to controlled conditions for degrading the 3xe2x80x2-end of the modified oligonucleotide thereby forming the oligonucleotide primer in a controlled manner for its extension along the polynucleotide template.
Another aspect of the present invention is a method for selectively extending an oligonucleotide primer along a specific target polynucleotide sequence in a mixture of polynucleotides. The method comprises providing in combination a mixture of polynucleotides and a modified oligonucleotide having a 3xe2x80x2-end that is not extendable along any polynucleotide and extending degraded modified oligonucleotide along the specific target polynucleotide sequence by controlling the degradation of the 3xe2x80x2-end of the modified oligonucleotide to give degraded modified oligonucleotide, which serves as an oligonucleotide primer in a chain extension reaction. In this way extension of the oligonucleotide primer along polynucleotides other than the specific target polynucleotide sequence is substantially reduced or avoided.
In another aspect the present invention provides an improvement in a method for amplifying a target polynucleotide sequence. The method comprises combining the target polynucleotide sequence with reagents for amplifying the target polynucleotide sequence and subjecting the combination to conditions wherein the target polynucleotide sequence is amplified. The reagents comprise a modified oligonucleotide and a polymerase. The improvement comprises the modified oligonucleotide having a portion that hybridizes to the target polynucleotide sequence except for the 3xe2x80x2-end thereof, which has at least one nucleotide analog that is incapable of hybridizing to a polynucleotide.
Another embodiment of the present invention is a method for amplifying a target polynucleotide sequence. A combination is provided that comprises the target polynucleotide sequence and reagents for conducting an amplification of the target polynucleotide sequence. The reagents comprise a polymerase, nucleoside triphosphates and a modified oligonucleotide, which comprises at least one nucleotide analog at the 3xe2x80x2-end thereof. The nucleotide analog is substantially non-hybridizable to any polynucleotide sequence and is also removable by a 3xe2x80x2 to 5xe2x80x2 exonuclease at a rate that is slower than for a natural nucleotide. The combination is subjected to conditions for amplifying the target polynucleotide sequence wherein the nucleotide analog is removed from the modified oligonucleotide. Extension of the oligonucleotide primer along the target polynucleotide sequence occurs to produce an extended oligonucleotide primer.
Another aspect of the present invention is a method for amplifying a target polynucleotide sequence in which the target polynucleotide sequence is combined with reagents for amplifying the target polynucleotide sequence and the combination is subjected to conditions to amplify the target polynucleotide sequence. The reagents comprise an oligonucleotide primer and a polymerase. The improvement comprises using a modified oligonucleotide having a portion that hybridizes to the target polynucleotide sequence except for the 3xe2x80x2-end thereof. The 3xe2x80x2-end is degradable at a rate of degradation that is slower for the modified oligonucleotide than for a corresponding oligonucleotide that does contain a modified nucleotide.
Another aspect of the present invention is an improvement in a method for forming multiple copies of a target polynucleotide sequence. A first oligonucleotide primer (xe2x80x9cfirst primerxe2x80x9d) is hybridized to the 3xe2x80x2-end of the target sequence. The first primer is extended along at least the target sequence in the presence of a polymerase. The first primer is capable of hybridizing to, and being extended along extended first primer or an extended second oligonucleotide primer (xe2x80x9csecond primerxe2x80x9d) that is different from the first primer. The extended second primer results from the extension of a second primer capable of hybridizing to and extending along a polynucleotide that is complementary (complementary polynucleotide) to the target sequence. Extended first primer is dissociated from the target sequence. The first or second primer is hybridized to the 3xe2x80x2-end of the extended first primer. The first or second primer is extended along the extended first primer and extended first primer or extended second primer is dissociated from extended first primer. The first primer is hybridized to the 3xe2x80x2-end of the extended first or second primer. Steps (e)-(g) are repeated. The improvement comprises using a modified oligonucleotide in place of the first oligonucleotide primer. The modified oligonucleotide has a portion that hybridizes to the target polynucleotide sequence except for the 3xe2x80x2-end thereof, which 3xe2x80x2-end has at least one nucleotide analog that is incapable of hybridizing to a polynucleotide.
Another embodiment of the present invention is a kit comprising in packaged combination (a) a modified oligonucleotide comprising at least one nucleotide analog at the 3xe2x80x2-end thereof, the nucleotide analog being substantially non-hybridizable to any normal nucleotide in a polynucleotide sequence, and (c) a 3xe2x80x2 to 5xe2x80x2 exonuclease. In one embodiment the nucleotide analog is removable by a 3xe2x80x2 to 5xe2x80x2 exonuclease at a rate that is slower than for a natural nucleotide, (b) nucleoside triphosphates